Enzymatically active eosinophil peroxidase associates with CD177 and alters oxidative activity in a subset of neutrophils

CD177 is a membrane protein present on some, but not all, circulating neutrophils in a given individual, dividing the total neutrophil population into two distinct subsets. We investigated proteomic differences between CD177 pos and CD177 neg neutrophils from healthy human subjects. Neutrophils were isolated from peripheral blood, followed by magnetic separation based on the surface expression of CD177. We performed tandem mass tag (TMT) using mass-spectrometry-based comparative proteomic analysis on ≥95% pure subpopulations, enabling reliable quantification of more than 3,400 proteins. Most proteins were similarly expressed in both neutrophil subsets, confirming a strong shared neutrophil identity. However, certain canonical eosinophil proteins, including eosinophil peroxidase (EPX), major basic protein (MBP), and MBP2 (PRG3), were upregulated in CD177 pos cells. Lysates of CD177 pos neutrophils showed higher peroxidase activity than CD177 neg neutrophils under conditions favoring EPX over myeloperoxidase, consistent with enzymatically active EPX, and flow cytometry showed surface localization of EPX on CD177 pos cells. Co-immunoprecipitation indicated that EPX associates with CD177. Our findings suggest that CD177 pos neutrophils exhibit a unique proteomic signature enriched in eosinophil-related proteins, and that EPX associated with CD177 may provide a neutrophil subset with altered enzymatic activity.